The long-term goal of this project is to understand how metallo intramembrane-cleaving proteases (MIPs) function in bacteria. MIPs are membrane-embedded enzymes that cleave their substrates within a membrane or near the membrane surface. Bacterial MIPs are known to play important roles during sporulation, stress responses, mating, polar morphogenesis, cell division, and infection. Understanding how MIPs function in bacteria could lead to the development of new antibiotics. In eukaryotes, MIPs cleave transcription factors that regulate lipid metabolism and the response to unfolded proteins in the endoplasmic reticulum. These pathways are critical for human health. Knowledge about bacterial MIPs will facilitate studies of eukaryotic MIPs, which could lead to the development of novel therapeutics. Little is known about how MIPs recognize their substrates or how MIP activity can be modulated. To fill this knowledge gap, most of the project focuses on SpoIVFB, which cleaves Pro-?K during Bacillus subtilis sporulation. The cleavage reaction has been reconstituted in vitro and requires ATP. Both ATP and Pro-?K bind to the CBS domain of SpoIVFB. CBS domains have been proposed to sense cellular energy levels and regulate the activity of a variety of proteins. The CBS domain of SpoIVFB may sense the energy level in the mother cell compartment of the sporangium and regulate access of Pro-?K to the active site of the enzyme. To test this model and to better understand how SpoIVFB recognizes Pro-?K, a combination of biochemical, structural, and genetic approaches is proposed. Likewise, a combination of approaches is proposed to achieve a molecular understanding of the mechanism of SpoIVFB inhibition by its natural inhibitor, the protein BofA. Knowledge from studies of SpoIVFB inhibition, substrate recognition, and the role of ATP could guide efforts to develop MIP modulators that benefit human health. B. subtilis codes for three other MIPs in addition to SpoIVFB. The most-studied of these, RasP, is representative of a subfamily of MIPs that is even more broadly conserved than the SpoIVFB subfamily, yet no biochemical studies on RasP have been reported. RasP subfamily members contain a PDZ domain and do not contain a CBS domain. Like certain other PDZ-domain-containing MIPs that have been studied, RasP functions in stress response and appears to cleave an anti-? transmembrane segment after initial cleavage of the anti-? extracytoplasmic domain. However, evidence suggests that RasP cleaves a cell division protein without a prior cleavage. Genetic and biochemical approaches are proposed to test this potential new paradigm. Neither of the known substrates of RasP accounts for certain defects of a rasP mutant or for the effects of RasP depletion. An innovative approach is proposed to identify the unknown substrate(s) of RasP. In addition to expanding knowledge of RasP, the approach could be used to identify substrates of other MIPs, overcoming a critical barrier to progress in the field. PUBLIC HEALTH RELEVANCE: Intramembrane-cleaving metalloproteases play important roles in bacteria when they infect humans, so these proteases are potential targets for the development of new antibiotics, and a closely related protease in humans functions in pathways critical for human health, making it a potential target for the development of novel therapeutics. The project aims to understand how intramembrane-cleaving metalloproteases function in bacteria, using the model organism Bacillus subtilis, which is closely related to several Bacillus species and Clostridia that cause disease, as well as to disease-causing bacteria such as Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumoniae. Knowledge about how intramembrane-cleaving metalloproteases of Bacillus subtilis function is expected to facilitate studies of these proteases in other bacteria, as well as studies of the human protease.